Stabilized formulation for luminescent detection of luciferase and nucleoside phosphates

ABSTRACT

Methods, kits and compositions containing a mixture of D-luciferin and L-luciferin for light generation with luciferase are disclosed that have improved stability when stored over time. The mixture of D-luciferin and L-luciferin can be used to detect the presence or amount of ATP or of luciferase in a sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional application of U.S. application Ser. No.14/186,065, filed Feb. 21, 2014, which claims priority to U.S.Provisional Application No. 61/767,875, filed Feb. 22, 2013, and U.S.Provisional Application No. 61/783,726, filed Mar. 14, 2013, all ofwhich are incorporated herein by reference in their entirety.

BACKGROUND

Luciferin is a racemic compound that, in solution, racemizes fromD-luciferin to L-luciferin or from L-luciferin to D-luciferin.D-luciferin can be utilized as a substrate by the enzyme luciferase togenerate light, whereas L-luciferin is largely inhibitory and causes adecrease in luminescence. Slow racemization of D-luciferin toL-luciferin during storage presents problems for assays that exploit theactivity of luciferase due to the varying and decreased availability ofD-luciferin substrate, and the inhibitory effects of L-luciferin.

FIELD

This disclosure relates to compositions, methods and kits useful forassaying enzymes and metabolites.

SUMMARY

In certain embodiments, a composition is provided comprising aluciferase, L-luciferin and D-luciferin, wherein the composition isessentially free of ATP. In certain embodiments, a composition isprovided comprising ATP, L-luciferin and D-luciferin, wherein thecomposition is essentially free of luciferase.

In some embodiments, a composition is provided comprising D-luciferin,L-luciferin and ATP.

In certain embodiments, a composition is provided comprising aluciferase, L-luciferin and D-luciferin and ATP in which theconcentration of L-luciferin exceeds the concentration of D-luciferin.

In some embodiments, a kit is provided which comprises a compositioncomprising a luciferase, L-luciferin and D-luciferin, the compositionbeing essentially free of ATP and packaged in a container. The kit caninclude at least one additional component such as one or more detergentssuch as dodecyltriammonium, hydroxypolyethoxydodecane, potassiumphosphate, a defoamer, a buffer, salt(s) and a chelator such asethylenediaminetetraacetic acid (EDTA).

In some embodiments, a kit is provided which comprises a compositioncomprising D-luciferin, L-luciferin and ATP, in which the concentrationof L-luciferin exceeds the concentration of D-luciferin. The kit caninclude at least one additional component such as one or more detergentssuch as dodecyltriammonium, hydroxypolyethoxydodecane, potassiumphosphate, a defoamer, a buffer, salt(s) and a chelator such asethylenediaminetetraacetic acid (EDTA).

In some embodiments, a kit is provided which comprises a compositioncomprising L-luciferin, D-luciferin and ATP the composition beingessentially free of luciferase and packaged in a container. The kit caninclude at least one additional component such as a buffer, a divalentcation chelator, a magnesium salt, an ionic or non-ionic detergent, athiol containing compound, such as coenzyme A or DTT, one or more sulfurcontaining reducing agents or a combination thereof.

In some embodiments, a method for determining the presence or amount ofATP in a sample is provided in which a composition comprisingluciferase, L-luciferin and D-luciferin is contacted with a sample, andthe luminescence generated is detected and/or measured to determine thepresence or amount of ATP in the sample.

In some embodiments, a method, kit or composition for determining thepresence or amount of luciferase in a sample is provided in which acomposition comprising D-luciferin, L-luciferin and ATP is contactedwith a sample which may contain a cell(s) expressing a luciferase. Theluminescence generated is then detected and/or measured to determine thepresence or amount of luciferase in the sample. The sample containing acell(s) expressing luciferase can be a lysed cell(s), a crude cellextract or a clarified cell extract.

In some embodiments, a method for detecting luciferase activity from acell expressing a luciferase is provided in which luciferase isexpressed in the cell, and the cell is contacted with a mixturecomprising L-luciferin and D-Luciferin at a ratio of L-Luciferin toD-Luciferin of at least about 5:95 to about 75:25. In some embodiments,the mixture comprises L-luciferin and D-Luciferin at a ratio ofL-Luciferin to D-Luciferin of at least about 5:95 to about 55:45, or atleast about 5:95 to about 50:50. The luminescence generated is thendetected and/or measured to determine the presence or amount ofluciferase in the cell.

In some embodiments, a method for detecting or quantifying a nucleosidephosphate such as ATP, or a source of ATP, in a cell or other sample isprovided in which the cell or sample is contacted with a mixturecomprising L-luciferin and D-Luciferin at a ratio of L-Luciferin toD-Luciferin of at least about 0.5:1 to about 2:1 and a luciferase. Theluminescence generated is then detected and/or measured to determine thepresence or amount of nucleoside phosphate in the cell.

In some embodiments, a method for performing a luciferase reaction isprovided. A mixture comprising L-luciferin and D-Luciferin is stored fora period of time at a temperature from about 0° C. to about 25° C. Whenthe mixture is used in a luciferase activity assay, the light output,i.e., luminescence, is measured. The mixture is permitted to becontacted with a sample, or part of a sample, containing or expressingluciferase, ATP or a source of ATP, to form a reaction assay. Theluminescence generated in the reaction assay is then detected. Thereaction assay containing a mixture that has been stored for a period oftime generates a light output that, in certain embodiments, is at leastabout 50% or at least about 90% of the light output of a comparablereaction assay containing the mixture at the start of the storage period(time t=0). In certain embodiments, the storage time period may be atleast about 30 minutes, at least about 1 hour, at least about 4 hours orlonger.

In some embodiments, a method for formulating components for aluciferase reaction is provided. D-Luciferin and L-luciferin arecombined to form a mixture, which is stored for a period of time, suchas a period of about 1, 2, 3 or 4 hours or 1 or 2 weeks at a temperaturefrom about 0° C. to about 25° C. The mixture allows for the generationof light output, i.e., luminescence, when used in a luciferase activityassay such that the light output generated after the period is at leastabout 50% or at least about 90% of the light output at the beginning ofthe period.

Other aspects of the invention will become apparent by consideration ofthe detailed description and accompanying drawings

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting luminescence detected from assays containingL-luciferin and D-luciferin after storage of the luciferin mixture atdifferent temperatures for different times.

FIG. 2 is a graph depicting luminescence detected from assays containingD-luciferin, or both D-luciferin and L-Luciferin after storage of theluciferin for various times.

FIG. 3 is a graph depicting luminescence detected from assays containingD-luciferin, or both D-luciferin and L-Luciferin after storage of theluciferin for various times.

FIG. 4 shows graphs depicting the effect of different D-/L-luciferin(5′-fluoroluciferin) ratios in luciferase reporter assay reagents onluminescence (4A) and signal half-life (4B).

FIG. 5 is a graph depicting the Effect of Different D-/L-luciferin(5′-fluoroluciferin) ratios on the stability of a luciferase reporterassay reagent.

DETAILED DESCRIPTION

Racemization of D-Luciferin to L-luciferin in a liquid formulationduring storage results in a mixture of the D- and L-enantiomers ofluciferin, of which D-luciferin can be utilized as a substrate by aluciferase enzyme to generate light. The unpredictability in the amountof D-luciferin available for luciferase is problematic for assays thatdepend upon measuring luciferase activity, such as those that measurethe amount of metabolites, e.g., ATP, in a sample. The presence ofL-luciferin in an assay may also be problematic as it acts as aninhibitor of the luciferase enzyme.

In some embodiments, a composition comprising both D-luciferin andL-luciferin provided in a mixture is disclosed. In some embodiments, thecomposition can be used with luciferase to measure the activity of anATP-utilizing enzyme, e.g., a kinase; to measure the amount orconcentration of ATP; to measure a particular metabolite in a samplethrough the intermediary production of ATP; to provide stablemeasurements over a period of time or a combination thereof. In someembodiments, the composition can be used to detect or measure the amountof luciferase expressed by a cell(s). Surprisingly, compositionscontaining a mixture of D-luciferin and L-luciferin show superiorstability over compositions containing substantially pure D-luciferin,or compositions comprising D-luciferin and substantially no L-luciferin,such as comprising trace amounts of L-luciferin, with minimal decreasein assay performance. For example, superior stability may occur whenL-luciferin is present in at least about 1%, in at least about 5%, in atleast about 10%, in at least about 15%, in at least about 25%, in atleast about 30%, in at least about 35%, at least about 40%, in at leastabout 45%, at least about 50%, in at least about 55%, at least about60%, in at least about 65%, at least about 70% or at least about 75% ormore of the total luciferin present, or when the amount of L-luciferinexceeds the amount of D-luciferin present. Metabolites that can bemeasured through the intermediary production of ATP include, withoutlimitation, nucleoside diphosphates such as adenosine diphosphate (ADP),guanosine diphosphate (GDP), uridine diphosphate (UDP), as well asadenosine monophosphate (AMP). In certain embodiments, the compositioncomprising the mixture of D-luciferin and L-luciferin optionallycomprises luciferase and may optionally be substantially free of ATP,other nucleoside triphosphates, nucleoside diphosphates, nucleosidemonophosphates or combinations thereof. In other embodiments, thecomposition comprising the mixture of D-luciferin and L-luciferin, forexample a mixture wherein the concentration of L-luciferin exceeds theconcentration of D-luciferin, may optionally comprise ATP.

D-luciferin includes the compound(D-(−)-2-(6′-hydroxy-2′-benzothiazolyl)-thiazoline-4-carboxylic acid),and L-luciferin includes the compound(L-(−)-2-(6′-hydroxy-2′-benzothiazolyl)-thiazoline-4-carboxylic acid)and their salt forms. As used herein, whether in D- or L-isomeric form,luciferin includes salt forms such as potassium salt, sodium salt, orother alkaline or alkaline earth salts, the free acid as well asluciferin derivatives and their salt forms such as chloroluciferin andfluoroluciferin e.g., 5′-fluoroluciferin, 7′-fluoroluciferin and5′-chloroluciferin and 7′-chloroluciferin and others disclosed in U.S.published application 2009-0075309, the entire disclosure of which isherein incorporated by reference.

D-Luciferin and L-luciferin (D-luciferin:L-luciferin) may be present ata ratio of least about 0.25:1, at least about 0.3:1, at least about0.4:1, at least about 0.5:1, at least about 0.6:1, at least about 0.7:1,at least about 0.8:1, at least about 0.9:1, at least about 1:1, and lessthan about 4:1, less than about 3.75:1, less than about 3.5:1, less thanabout 3.25:1, less than about 3:1, less than about 2.75:1, less thanabout 2.5:1, less than about 2.25:1, less than about 2:1, less thanabout 1.9:1, less than about 1.8:1, less than about 1.7:1, less thanabout 1.6:1, less than about 1.5:1, less than about 1.4:1, less thanabout 1.3:1, less than about 1.25:1, less than about 1.2:1 or less thanabout less than about 1.1:1.

L-Luciferin and D-luciferin (L-luciferin:D-luciferin) may be present ata ratio of at least about 0.1:99.9, least about 1:99, at least about2:98, at least about 3:97, at least about 4:96, at least about 5:95, atleast about 10:90, at least about 15:85, at least about 20:80, at leastabout 25:75, at least about 30:70, at least about 35:65, at least about40:60, at least about 45:55, at least about 49:51, or at least about50:50, and less than about 99:0.1, less than about 99:1, less than about98:2, less than about 97:3, less than about 96:4, less than about 95:5,less than about 90:10, less than about 85:15, less than about 80:20,less than about 75:25, less than about, 70:30, less than about 65:35,less than about 60:40, less than about 55:45, less than about 51:49, orless than about 50:50.

The mixture comprising D-luciferin, L-luciferin and optionally aluciferase may be substantially free of or exclude any amount ofnucleoside triphosphates, nucleoside diphosphates or any combinationthereof. For example, the mixture comprising D-luciferin, L-luciferinand optionally a luciferase may be substantially free or exclude anyamount of ATP, GTP, CTP, mSUTP, UTP or combination thereof. The mixturecomprising D-luciferin, L-luciferin and optionally a luciferase may besubstantially free or exclude any amount of ADP, GDP, CDP, m5UDP, UDP orcombination thereof. The mixture comprising D-luciferin, L-luciferin andoptionally luciferase may contain less than about 50 μM ATP, less thanabout 10 μM ATP, less than about 5 μM ATP, less than about 1 μM ATP,less than about 0.5 μM ATP, less than about 0.1 μM ATP, less than about0.05 μM ATP, less than about 0.01 μM ATP, less than about 0.005 μM ATP,or less than about 0.001 μM ATP, or less than about 0.0001 μM ATP orother nucleoside triphosphate described herein.

In some embodiments, the mixture comprising D-luciferin, L-luciferin andoptionally luciferase may contain at least 0.005 μM ATP, at least about0.01 ATP, at least about 0.05 μM ATP, at least about 0.1 μM ATP, atleast about 0.5 μM ATP, at least about 1 μM ATP, at least about 5 μMATP, at least about 10 μM μM ATP, at least about 5004 ATP and less thanabout 10 mM ATP, less than about 5 mM ATP, less than about 4 mM ATP,less than about 3 mM ATP, less than about 2 mM ATP, less than about 1 mMATP, less than about 0.5 mM ATP. For example, such a mixture maycomprise a ratio of L-luciferin:D-luciferin that is greater than 1:1,greater than 1.1:1, greater than 1.2:1, greater than 1.3:1, greater than1.4:1, greater than 1.5:1, greater than 1.6:1, greater than 1.7:1,greater than 1.8:1, greater than 1.9:1, greater than 2:1, greater than2.25:1, greater than 2.5:1, greater than 2.75:1, or greater than 3:1.

The mixture comprising D-luciferin and L-luciferin may optionallysubstantially include any amount of nucleoside triphosphates, e.g., ATP,GTP, CTP, nucleoside diphosphates, e.g., ADP, GDP, CDP or anycombination thereof.

The mixture comprising D-luciferin and L-luciferin remains stable overlong periods of time, where stability is measured by light output whenthe mixture is used in a luciferase activity assay. In contrast toconventional formulations, which can show about a 10% loss of activityafter 8 hours at 22° C. or after 4 days at 4° C., the presentformulations exhibit increased stability. At least about 50%, at leastabout 55%, at least about 60%, at least about 65%, at least about 70%,at least about 75%, at least about 80%, at least about 85%, at leastabout 90%, or at least about 95% of the original activity (whereoriginal activity is measured in a luciferase activity assay at T=0under a given set of conditions and the subsequent activity is measuredunder substantially the same conditions at a later time point) isdetected after a period of storage of at least about at least about 8hours, at least about 10 hours, at least about 12 hours, at least about18 hours, at least about 1 day, at least about 2 days, at least about 3days, at least about 4 days, at least about 5 days, at least about 1week, at least about 2 weeks, at least about 3 weeks, at least about 4weeks, at least about 1 month, at least about 2 months, at least about 3months, at least about 4 months, at least about 5 months, at least about6 months, at least about 9 months, at least about 12 months, at leastabout 18 months, at least about 2 years, at least about 3 years, atleast about 4 years or longer, when stored, for example, at atemperature of about 4° C. (for example from about 1° C. to about 6° C.)or about 20° C. to 25° C., such as about 22° C. (for example from about20° C. to about 25° C.). For example, methods, compositions and kitsdescribed herein using about a 50:50 mixture of L-luciferin andD-luciferin will have at least about 50% of the original activityremaining after incubation of about or at least about 2 months at atemperature from about 20° C. to about 25° C.

A luciferase activity assay includes, for example, luciferase, luciferin(e.g. 1 mM), ATP (e.g., 3 mM), MgSO₄ (e.g., 15 mM) in buffer at a pHthat is optimal for the enzyme used. Other optional components may bepresent to optimize or stabilize the activity of the luciferase used.Examples of luciferase activity assays are described herein in theexamples. In some embodiments, the luciferase may be purified, or it maybe expressed in the sample, e.g., a reporter gene.

Kits, methods and compositions comprising a mixture of D-luciferin andL-luciferin are provided herein. The kits, methods and compositions mayinclude one or more luciferases or may be used to assay luciferase in atransformed prokaryotic or transfected eukaryotic cell using aluciferase reporter assay. The cell can be, for example, a bacterialcell, a mammalian cell, a plant cell or a fungal cell.

Luciferases, which utilize luciferin as a substrate, include beetleluciferases, such as that of the common firefly (family Lampyridae).Beetle luciferases are often referred to as firefly luciferases in theliterature; however, firefly luciferases are actually a subgroup of thebeetle luciferase class. Beetle luciferases also include click beetleluciferase, such as from Pyrophorus plagiophthalamus. Beetle luciferasesmay be purified from the lanterns of the beetles themselves or fromprotein expression systems well known in the art.

Beetle luciferases, particularly firefly luciferase from the NorthAmerican firefly Photinus pyralis or Photuris pennsylvanica, are wellknown in the art. The P. pyralis luciferase (LucPpy) consists ofapproximately 550 amino acids (61 kDa) as calculated by the proteinencoded by the nucleotide sequence of the gene. The Photurispennsylvanica firefly luciferase (LucPpe2) consists of 545 amino acidresidues (GenBank 2190534; Ye et al., 1997). Mutant luciferases, e.g.,thermostable and/or chemostable, derived from LucPpe2 may includeLucPpe2m78 (also known as 78-0B10), LucPpe2m90 (also known as 90-1B5),LucPpe2m133 (also known as 133-1B2) and LucPpe2m146 (also known as146-1H2). However, any luciferase that meets the limitations set forthherein may be used in the composition, method and kits of the invention.A method of deriving thermostable and/or chemostable luciferases such asLucPpe2m78, LucPpe2m90, LucPpe2m133, and LucPpe2m146 is disclosed inPCT/US99/30925.

Isolated and/or purified luciferases can be used. Contaminant componentsof the luciferase natural environment are materials that would typicallyinterfere with the luciferase assay, and may include enzymes, hormones,and other proteinaceous or non-proteinaceous materials. One technique toascertain purity is applying SDS-PAGE analysis under non-reducing orreducing conditions using Coomassie blue or silver stain.

Luciferases useful in the compositions, kits and methods of theinvention include those that generate a stable signal and/or arethermostable and/or chemostable, i.e., they yield enhanced duration ofluminescence in a luciferase reaction defined as less than 50% loss ofluminescence per 30 minutes relative to the luminescence at the time theluciferase reaction was initiated or greater enzyme stability at highertemperatures. Exemplary luciferases are disclosed in U.S. Pat. Nos.6,132,983; 6,171,808; 6,265,177; 6,602,677; 7,241,584; 7,906,298; and8,030,017; and U.S. Patent Application Publication Nos. 2003-0068801,2006-0183212, 2009-0137019, 2011-0177540, and 2012-0009647, thedisclosure of each of which is herein incorporated by reference in itsentirety.

Luciferases in the compositions, kits and methods of the invention alsoinclude luciferases that generate a “flash” signal and have a half-lifefor light output of less than about 30 minutes, less than about 25minutes, less than about 20 minutes, less than about 15 minutes, lessthan about 10 minutes, less than about 5 minutes, less than about 4minutes, less than about 3 minutes, less than about 2 minutes, or lessthan about 1 minutes.

Luciferases include those that display increased thermostability for atleast 2 hours at 50° C., or at least 5 hours at 50° C. Thermostableluciferases which, when solubilized in a suitable aqueous solution,include those having a stability half-life greater than about 2 hours atabout 50° C., greater than about 5 hours at 50° C., greater than about10 hours at 50° C., greater than about 5 hours at about 60° C., greaterthan about 10 hours at about 60° C., greater than about 24 hours atabout 60° C., greater than about 3 months at about 22° C. or greaterthan about 6 months at 22° C.

In certain embodiments, the amount of luciferase present in a reactionmixture comprising a racemic mixture of L-luciferin and D-luciferin, asdescribed herein, may be increased to provide a light output that issubstantially equivalent to a similar reaction mixture that does notcomprise a racemic mixture of L-luciferin and D-luciferin. For example,the amount of luciferase may be increased to at least about 120%, atleast about 150%, at least about 200% (2-fold), at least about 250%, atleast about 300%, at least about 350%, at least about 400%, at leastabout 500% (5-fold), at least about 600%, at least about 700%, at leastabout 800%, at least about 900% or at least about 1000% (10-fold) of theluciferase amount in the reaction mixture that does not comprise aracemic mixture of L-luciferin and D-luciferin to achieve asubstantially equivalent light output.

In certain embodiments, the compositions, methods and kits describedherein provide an increased half-life of the luminescent signalgenerated during a luciferase reaction. The compositions, methods andkits described herein containing a mixture of D-luciferin andL-luciferin may increase the half-life of the luminescent signal(luminescence generated during a luciferase reaction) by at least about5%, at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, at least about 100%, at least about 125%, at least about150%, at least about 175%, at least about 200%, at least about 250%, atleast about 300%, or at least about 350%, at least about 400%, at leastabout 450%, or at least about 500%, compared with the luminescent signalgenerated from a luciferase reaction that comprises substantially pureD-luciferin, or D-luciferin and substantially no L-luciferin, such ascomprising trace amounts of L-luciferin. In some embodiments at leastabout a 2-fold, at least about a 3-fold, at least about a 4-fold, atleast about a 5-fold, at least about a 6-fold, at least about a 7-fold,at least about a 8-fold, at least about a 9-fold, at least about a10-fold, at least about a 12-fold, at least about a 15-fold, or at leastabout a 20-fold increase in luminescent signal half-life is achievedcompared with the luminescent signal generated from a luciferasereaction that comprises substantially pure D-luciferin, or D-luciferinand substantially no L-luciferin, such as comprising trace amounts ofL-luciferin.

The light output can be measured in relative light units (RLUs). In someembodiments, the initial light output of the luminogenic reaction islower when a mixture of D-luciferin and L-luciferin is used. Forexample, the initial light output may be less than about 95%, less thanabout 90%, less than about 85%, less than about 80%, less than about75%, less than about 70%, less than about 65%, less than about 60%, lessthan about 55%, less than about 50%, less than about 45%, less thanabout 40%, less than about 35%, less than about 30%, less than about25%, less than about 20%, less than about 15%, less than about 10%, orless than about 5% of the initial light output compared with aluminogenic reaction that comprises substantially pure D-luciferin, orD-luciferin and substantially no L-luciferin, such as comprising traceamounts of L-luciferin. In some embodiments, the lower initial lightoutput is accompanied by an increased half-life of the luminogenicreaction, as described herein.

In kits, methods and compositions described herein, luciferase may beprovided in a solution, such as an aqueous solution, containingL-luciferin and D-luciferin, or may be provided in a desiccated or driedform, for example, in a container separate from the D-luciferin andL-luciferin, which is solubilized prior to use. In some embodiments,luciferase is provided in a container separate from the D-luciferin andL-luciferin in solubilized form. The amount of luciferin, whetherD-luciferin, L-luciferin or a combination of D-luciferin andL-luciferin, included in the compositions, kits and methods describedherein can be at least about 1 nM, at least about 5 nM, at least about10 nM at least about 50 nM at least about 100 nM, at least about 0.5 μM,at least about 1 μM, at least about 5 μM, at least about 10 μM, at leastabout 25 μM, at least about 50 μM, at least about 100 μM, at least about250 μM, at least about 0.5 mM, at least about 1 mM, and less than about50 mM, less than about 40 mM, less than about 30 mM, less than about 30mM, less than about 10 mM, less than about 5 mM, less than about 4 mM,less than about 3 mM, or less than about 2 mM.

Kits, compositions and methods described here may include, in additionto a mixture of D-luciferin and L-luciferin described herein, one ormore additional components, such as luciferase; a buffer such as citricacid or citrate buffer, MES, 1,4-Piperazinediethanesulfonic acid, orHEPES; inorganic phosphate, for example, in the form pyrophosphate orpotassium phosphate; a chelator such as EDTA, CDTA or1,2-Diaminocyclohexanetetraacetic acid; a salt such as sodium fluoride,magnesium sulfate; a surfactant or detergent such as TERGITOL® (e.g. anon-ionic nonylphenol ethoxylate), dodecyltrimethylammonium bromide(DTAB) or THESIT® (hydroxypolyethoxydodecane); a defoamer such asINDUSTROL® DF204 (organic defoamer) or MAZU® DF (silicone defoamer); aprotein stabilizer such as gelatin, PRIONEX® 10% (gelatin, Type A) oralbumin (e.g. BSA, HSA) or glycerol; adenosine triphosphate (ATP) oradenosine monophosphate (AMP). Other components may include polyethyleneglycol, polyvinyl pyridine, crown ether, or cyclodextrin. Additionalcomponents may include, for example, one or more of, thiol compounds,such as coenzyme A, reducing agents such as dithiothreitol (DTT), orsulfur containing compounds acting as reductants, such as sulfite,thiosulfate.

Detergents that may be included in the kits, compositions and methodsinclude cationic, anionic, non-ionic or zwitterionic detergents. Thedetergent can comprise, for example, Tergitol® detergent (polyglycolether (nonionic)), Brij 35® detergent (polyoxyethylene 23 lauryl ether),Brij 58® detergent (polyoxyethylene 20 cetyl ether(HO(CH₂CH₂O)₂₀C₁₆H₃₃)), Triton X-100® detergent(4-(1,3,3-tetramethylbutyl)phenyl-polyethylene glycol(t-Oct-C₆H₄—(OCH₂CH₂)_(x)OH, x=9-10)), Triton X-305® detergent(4)-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol), Triton N101®detergent (polyoxyethylene-9,10 branched nonylphenyl ether), CHAPS®detergent (3[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate),Chapso® detergent(3-([3-cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate),Bigchap® detergent (N,N-bis(3-D-Gluconamidopropyl)cholamide), Thesit®detergent (polyethylene glycol 400 dodecyl ether(HO(CH₂CH₂O)_(n)(CH₂)₁₁CH₃)), Pluronic L64® detergent (poly(ethyleneglycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)),Rhodasurf 870® detergent (polyethoxylated (20) oleyl alcohol), ChemalLA-9® detergent (polyoxyethylene 9 lauryl alcohol), Sulfonyl 465®detergent (2,4,7,9-tetramethyl-5-deceyne-4,7-diol ethoxylate 10),deoxycholate, CTA 3, Pierce C08® detergent(C8=Octyl-β-D-glucopyranoside), or Pierce C100 detergent(n-decyl-.beta.-D-maltoside (C10 alkyl side chain)).

The mixture containing D-luciferin and L-luciferin used in the methods,kits and compositions described herein or the assays described hereinwhich utilize the D-luciferin and L-luciferin can have a pH of at leastabout 5, at least about 5.1, at least about 5.2, at least about 5.3., atleast about 5.4, at least about 5.5, at least about 5.6, at least about5.7, at least about 5.8, at least about 5.9, at least about 6, at leastabout 6.1, at least about 6.2, at least about 6.3, at least about 6.4,at least about 6.5, at least about 6.6, at least about 6.7, at leastabout 6.8, at least about 6.9, at least about 7, at least about 7.1, atleast about 7.2, at least about 7.3, at least about 7.4, at least about7.5, at least about 7.6, at least about 7.7, at least about 7.8, atleast about 7.9, at least about 8, at least about 8.1, at least about8.2, at least about 8.3, at least about 8.4, or at least about 8.5 andless than about 9, less than about 8.9, less than about 8.8, less thanabout 8.7, less than about 8.6, less than about 8.5, less than about8.4, less than about 8.3, less than about 8.2, less than about 8.1, lessthan about 8, less than about 7.9, less than about 7.8, less than about7.7, less than about 7.6, less than about 7.5, less than about 7.4, lessthan about 7.3, less than about 7.2, less than about 7.1, less thanabout 7, less than about 6.9, less than about 6.8, less than about 6.7,less than about 6.6, or less than about 6.5. The pH can be maintained inthe mixture composition or assay to provide an environment thatfacilitates bright luminescence at a fast rate, such as a pH of fromabout 8 to 9, or a slower rate of luminescence providing more sustainedluminescence, such as a pH of less than about 7.5 or less than about 7.

The D-Luciferin and L-luciferin may be combined together at any of theratios described herein prior to being stored for a period of time.Luciferase, ATP, or any other component of the kits or compositionsdisclosed herein, may be optionally combined with a mixture ofD-luciferin and L-luciferin prior to storage. The mixture may be storedat a temperature of at least about −85° C., at least about −80° C., atleast about −75° C., at least about −50° C., at least about −40° C., atleast about −30° C., at least about −25° C., at least about −20° C., atleast about 0° C., at least about 1° C., at least about 2° C., at leastabout 3° C. or at least about 4° C. and less than about 45° C., lessthan about 40° C., less than about 38° C., less than about 35° C., lessthan about 30° C., less than about 25° C., less than about 22° C., lessthan about 21° C., less than about 20° C., less than about 15° C., lessthan about 10° C., less than about 8° C., or less than about 6° C. for atime period. The time period for storage may be at least about 4 hours,at least about 8 hours, at least about 12 hours, at least about 1 day,at least about 1 week, at least about 2 weeks, at least about 4 weeks,at least about 2 months, at least about 3 months, at least about 4months, at least about 6 months, or at least about 12 months. The amountof D-luciferin remaining after the period of storage may be at leastabout 99%, at least about 98%, at least about 95%, at least about 90%,at least about 85%, at least about 80%, at least about 75%, or at leastabout 70%, at least about 65%, at least about 60%, at least about 55%,at least about 54%, at least about 53%, at least about 52%, or at leastabout 51% of the initial amount of D-luciferin in the mixture prior tostorage. The amount of D-luciferin remaining after the period of storagemay be at least about 101%, at least about 105%, at least about 110%, atleast about 115%, at least about 120%, at least about 125%, at leastabout 130%, at least about 140%, at least about 150%, at least about175%, at least about 200%, at least about 250%, at least about 300%, atleast about 400%, at least about 500%, at least about 750%, at leastabout 1000%, of the initial amount of D-luciferin in the mixture priorto storage, for example, if the formulation comprises more L-luciferinthan D-luciferin prior to storage.

The compositions and kits disclosed herein may be used to assay thepresence or amount of ATP in a sample by a luciferase-luciferin reactionwhich generates luminescence. The ATP may be present in the samplefollowing an ATP-utilizing reaction, e.g., kinase reaction, such thatthe amount of ATP in the sample and luminescence generated is inverselyproportional to the amount of activity of the ATP-utilizing enzyme. Thesample may contain intact cells, may be a crude or clarified cell lysateor may comprise a purified enzyme which generates or uses ATP.

The compositions and kits disclosed herein may be used to assay thepresence or amount of luciferase in a sample. The luciferase may bepresent in the sample following transfection, e.g., transient or stable,of a luciferase gene, e.g., a reporter gene. The sample may containintact cells, crude or clarified cell lysate or comprise a whole animal,such as for whole animal imaging. The luciferase may be present in thesample following expression of the luciferase coding sequence incell-free extract, e.g., rabbit reticulocyte lysate or wheat germtranslation systems.

It is to be understood that the invention is not limited in itsapplication to the details of construction and the arrangement ofcomponents set forth in the description or illustrated in the followingdrawings. The invention is capable of other embodiments and of beingpracticed or of being carried out in various ways.

It is to be understood that any numerical range recited in thisspecification includes all values from the lower value to the uppervalue. For example, if a concentration range is stated as 1% to 50%, itis intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%,etc., are expressly enumerated in this specification. It is also to beunderstood that any numerical range recited in this specificationincludes all values from at least the lower value without an upperlimit, and all values up to the upper value without a lower limit. Theseare only examples of what is specifically intended, and all possiblecombinations of numerical values between and including the lowest valueand the highest value enumerated are to be considered to be expresslystated in this application.

It also is to be understood that the invention is not limited in itsapplication to the details of construction and the arrangement ofcomponents set forth in the description. Also, it is to be understoodthat the phraseology and terminology used in this specification is forthe purpose of description and should not be regarded as limiting. Theuse of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated in thisspecification or clearly contradicted by context. The terms“comprising,” “having,” “including,” and “containing” are to beconstrued as open-ended terms (i.e., meaning “including but not limitedto”) unless otherwise noted. All methods described in this specificationcan be performed in any suitable order unless otherwise indicated inthis specification or otherwise clearly contradicted by context. Patentapplications, patents and literature references cited here arespecifically and completely incorporated by reference in their entirety.Where inconsistent interpretations are possible, the present disclosurecontrols.

The use of any and all examples, or exemplary language (e.g., “such as”)provided here, is intended merely to illustrate aspects and embodimentsof the disclosure and does not limit the scope of the claims.

Example 1: Accelerated Stability

5 mM L-luciferin was dissolved in CellTiter-Glo (an ATP detectionreagent containing Ultra-Glo luciferase and 5 mM D-luciferin; PromegaCorporation). Aliquots were placed at various temperatures for variouslengths of time and then stored at −80° C. All samples were thawed toroom temp at the same time and then mixed 1:1 with 1 μM ATP in water.Luminescence (RLUs) was measured after 10 min. The results are depictedin FIG. 1.

From the decay rates at higher temperature, the Arrhenius equation wasused to calculate the rates of decay at lower temperatures. At 22° C., a10% loss in performance was calculated to occur in 8.6 days. At 4° C., a10% and 50% loss at 4.2 months and 2 years, respectively, wascalculated. At −20° C., a 10% loss in 20 years was calculated.

Example 2: Real-time Stability at 22° C.

Aliquots of CellTiter-Glo with and without 5 mM L-luciferin were placedat 22° C. for various lengths of time and then stored at −80° C. Allsamples were thawed to room temp at the same time and then mixed 1:1with 1 μM ATP in water. Luminescence (RLUs) was measured after 10 min.The results are depicted in FIG. 2.

At 22° C., a 10% change in performance occurred in about 12 hours orabout 2 weeks for CellTiter-Glo without and with L-luciferin,respectively.

Example 3—Comparison of Luciferin and 5′-Fluoroluciferin

5 mM L-isomer luciferin or fluoroluciferin was dissolved in a reagent(500 mM MES, pH6, 400 mM KCl, 3 mM CDTA, 20 mM MgSO4, 2 mM NaF, 15 μMtetrasodium pyrophosphate, 2% Thesit, 1% DTAB, 0.2% Mazu, and 0.21 mg/mlUltra-Glo) containing 5 mM of the respective D-isomer. Each reagent,including CellTiter-Glo, was then mixed 1:1 with 2 μM ATP in water, andluminescence (RLUs) measured over time. The results are depicted inTable 1.

TABLE 1 luciferin 5′-fluoro RLU @ 10′ half-life (hrs) D 31,685,833 1.72D + L 7,229,377 6.66 D 27,391,967 2.75 D + L 12,016,733 5.56 CTG5,181,023 5.25

Although there is only a modest difference in light output whencomparing the D-isomers, the isomeric mixture of 5′-fluoroluciferin issubstantially brighter than that of luciferin with roughly the samesignal half-life.

Example 4. Enhanced Stability in a Reagent Containing Native FireflyLuciferase

1 mM L-luciferin was dissolved in a reagent (50 mM MgAcetate, 25 mMTrisAcetate, pH 7.75, 0.1 mM EDTA, 0.002% azide, 0.5 mM DTT, 1.3 mg/mlBSA) containing 1 mM D-luciferin and 13.54 μg/ml native fireflyluciferase (QuantiLum® recombinant luciferase, Promega, Madison Wis.).Aliquots were placed at 22° C. for various lengths of time and thenstored at −80° C. All samples were thawed to room temp at the same timeand then mixed 1:1 via injection with 20 nM ATP in water. After a 10second delay, luminescence (RLUs) was measured and integrated for 10seconds. The results are depicted in FIG. 3.

At 22° C., a 10% change in performance occurred in about 5 hours andabout 50 hours with reagents without or with the L-isomer of luciferin,respectively.

Example 5. Effect of Different D-/L-luciferin Ratios in LuciferaseReporter Assay Reagent on Luminescence and Signal Half-Life

HEK293 cells stably expressing firefly luciferase (CMV-Fluc) were platedin white 96-well plates at 2.5×10⁵ cells/ml in 100 μl complete media(DMEM+10% FBS+1×NEAA (non-essential amino acid)). The cells were thenincubated overnight at 37° C., 5% CO₂. After the overnight incubation,the plate was equilibrated to 22° C. 100 μl of firefly luciferasereporter assay reagents containing 1 mM luciferin at 100:0; 95:5; 90:10;85:15; 80:20; or 75:25 D-/L-5′-fluoroluciferin were added to the cells(n=3) and incubated with mixing for 3 minutes. Luminescence was measuredevery 3 minutes for 5 hours on a GloMax® Multi+ at 22° C.

FIG. 4 demonstrates the effect of the different D-/L-luciferin ratios oninitial luminescence (RLUs) (A) and signal half-life (B). The signalhalf-life was calculated by fitting the luminescence to a singleexponential decay.

Example 6. Effect of Different D-/L-luciferin Ratios on the Stability ofa Luciferase Reporter Assay Reagent

Firefly luciferase reporter assay reagents were made with varying ratiosof D-/L-5′-fluoroluciferin (100:0; 90:10; 80:20; 70:30; 60:40; and50:50). Each reagent was dispensed into 180 μl aliquots and incubated ina 22° C. water bath for varying amounts of time (0, 1, 2, 3, 4 or 5days). After each time period, an aliquot of reagent with each ratio wasfrozen at −80° C. After the time course was finished, all aliquots werethawed, mixed by pipetting up and down, and 50 μl transferred totriplicate wells of a 96-well plate, which was then equilibrated at 22°C. Purified firefly luciferase (QuantiLum) was diluted to 0.69 μg/ml inDMEM supplemented with 0.1% Prionex, and 50 μl was added to each welland mixed for 3 minutes. Luminescence was read on a GloMax®Multi+luminometer.

FIG. 5 shows enhanced room temperature stability of the luciferasereporter assay reagent as the ratio of D-/L-5′fluoroluciferin getscloser to 50:50.

1.-21. (canceled)
 22. A composition comprising a combination ofD-luciferin and L-luciferin present in the composition in a ratio fromabout 0.25:1 to about 4:1 of D-luciferin to L-luciferin; wherein thecomposition is substantially free of adenosine triphosphate (ATP); andwherein the composition retains at least 50% luciferase activity afterbeing stored for at least about 8 hours at a temperature from at leastabout −85° C. to about 45° C.
 23. The composition of claim 22, whereinthe composition retains at least 50% luciferase activity after beingstored for at least about 8 hours at a temperature from about 0° C. toabout 35° C. as compared to a composition that has substantially noL-luciferin.
 24. The composition of claim 22, wherein the ratio ofD-luciferin to L-luciferin is from about 0.5:1 to about 2:1.
 25. Thecomposition of claim 22, wherein the ratio of D-luciferin to L-luciferinis from about 1:1 to about 4:1.
 26. The composition of claim 22, whereinthe ratio of D-luciferin to L-luciferin is about 1:1.
 27. Thecomposition of claim 22, wherein the composition has a pH of at leastabout 5 and less than about
 9. 28. The composition of claim 22, furthercomprising a luciferase.
 29. The composition of claim 28, wherein theluciferase is a beetle luciferase.
 30. The composition of claim 22,wherein the D-Luciferin is D-5′fluoroluciferin and wherein theL-luciferin is L-5′fluoroluciferin.
 31. The composition of claim 22,wherein the composition retains at least 50% luciferase activity afterbeing stored for at least about 8 hours at a temperature from about 0°C. to about 35° C.
 32. The composition of claim 22, wherein thecomposition retains at least 50% luciferase activity after being storedfor at least about 8 hours at a temperature from about 20° C. to about35° C.
 33. The composition of claim 22, wherein the composition retainsat least 50% luciferase activity after being stored for at least about 8hours at a temperature from about 20° C. to about 30° C.
 34. Thecomposition of claim 22, wherein the composition retains at least 50%luciferase activity after being stored for at least about 10 hours at atemperature from about −85° C. to about 45° C.
 35. The composition ofclaim 22, wherein the composition retains at least 50% luciferaseactivity after being stored for at least about 12 hours at a temperaturefrom about −85° C. to about 45° C.
 36. The composition of claim 22,wherein the composition retains at least 50% luciferase activity afterbeing stored for at least about 18 hours at a temperature from about−85° C. to about 45° C.
 37. The composition of claim 22, wherein thecomposition further comprises at least one additional component selectedfrom: a buffer, protein stabilizer, magnesium ions, a metal chelator, adetergent, a defoamer, inorganic phosphate, pyrophosphate, AMP, coenzymeA, a reducing agent, or a combination thereof.
 38. The composition ofclaim 22, wherein the composition comprises magnesium sulfate andtrans-1,2-Cyclohexanediaminetetraacetic Acid (CDTA).
 39. The compositionof claim 22, wherein the composition further comprises at least oneadditional component selected from: 1,4-Piperazinediethanesulfonic acid,1,2-Diaminocyclohexanetetraacetic acid, Tergitol detergent, Mazudefoamer, pyrophosphate, DTT, sulfite, thiosulfate, sodium fluoride,dodecyltrimethylammonium, hydroxypolyethoxydodecane, potassiumphosphate, ethylenediaminetetraacetic acid (EDTA), or a combinationthereof.
 40. A method for determining the presence or amount of ATP in asample comprising contacting the composition of claim 1 with a sampleand detecting luminescence generated, thereby determining the presenceor amount of ATP in the sample.
 41. The method of claim 40, wherein thesample comprises intact cells and/or a purified enzyme which generatesor uses ATP.
 42. The method of claim 40, wherein the sample comprises acrude cell lysate or a clarified cell lysate.